Selenophosphate (SeP), shown earlier to be the selenium donor in prokaryotes for selenoprotein and seleno-tRNA biosynthesis, is an oxygen- labile compound formed from selenide and ATP by selenophosphate synthetase. To detect an enzyme-bound pyrophosphate group, the putative intermediate in this reaction, labeling experiments using [gamma-32P]ATP, [beta-32P]ATP, [14C]ATP, and [75Se]selenide were carried out. Although the gamma phosphoryl group of ATP was bound to enzyme, the beta phosphoryl group was not detected showing that no stable enzyme- pyrophosphate intermediate existed. The additional presence of [14C]AMP and [75Se] on the isolated enzyme suggests that two of the final products, AMP and SeP (derived from the gamma phosphoryl group) remained bound to protein. Pi, the third product, which is derived from the beta phosphoryl group, was not retained. Detection and purification of selenophosphate synthetase from mammalian and Archae sources shows that SeP is a general biological Se donor. Structural differences in the enzymes from these sources are of potential use in mechanism studies. Although a murine embryonic selenophosphate synthetase gene contains a TGA codon, the enzyme isolated from Methanococcus vannielii does not contain selenium. A new mammalian selenoprotein, thioredoxin reductase, was isolated from a human lung adenocarcinoma cell line. The amount of enzyme in two transformed cell lines, lung cells and HeLa cells, was 5-to-10-fold higher than reported previously in normal mammalian tissues. Selenium was shown to be present in the form of a selenocysteine residue in a position corresponding to TGA in the human placental gene. The C- terminal sequence is -Cys-SeCys-Gly. Two forms of the protein, differing in ability to bind to heparin affinity columns and to interact with rat liver thioredoxin reductase polyclonal antibodies, were isolated from 75Se-labeled human lung and HeLa cells. These proteins were indistinguishable by SDS PAGE, catalytic activity, FAD, and selenocysteine content and C-terminal region amino acid sequence. M. vannielii cells labeled with 75Se in the presence of urate contain an unidentified 33 kDa selenoprotein that has been purified and partially sequenced. By products of the isolation procedure are 75Se-labeled formate dehydrogenase and hydrogenase. Since these multimeric proteins contain subunits similar in size to the unknown protein, protein bands from SDS PAGE gels of each will be recovered and subjected to amino acid sequence analysis for comparison.